This invention relates to methods for treating patients having deficiencies or inhibitors of blood clotting factors, particularly factor VIII (antihemophilic factor, AHF).
Blood coagulation is an exceedingly complex process. The interaction of various blood components which eventually gives rise to a fibrin clot has been compared to a cascade of steps, each of which is dependent upon and regulated by preceding and following steps. Generally, the blood components which take part in the coagulation cascade are either proenzymes or enzyme modulators. The proenzymes are enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an "activator", generally another proteolytic enzyme produced at an earlier stage in the coagulation cascade. Coagulation factors which have undergone such a conversion are hereafter defined as activated factors, and designated by the lower case postscript "a" while the proenzymes are referred to as precursor clotting factors.
The enzyme modulators are principally cofactors such as calcium ions or nonenzyme proteins and most are essential if the enzymes are to exhibit any catalytic activity at all. Such modulators are to be distinguished from enzyme substrates. Substrates are compounds which are covalently modified by an enzyme while modulators or cofactors merely bind to the enzyme without undergoing a change in structure.
Proenzymes are key components in the blood coagulation cascade. They constitute a reservoir to supply the needs of the clotting cascade, being activated to the enzymatically functional form as required. The extent of activation and the activity of the enzymes are controlled by the modulators.
Factor VII is one of these proenzymes. Factor VII participates in the extrinsic pathway of blood coagulation by converting factor X to Xa in the presence of tissue factor. Factor Xa in turn then converts prothrombin to thrombin in the presence of the cofactors factor V, calcium ions and phospholipid. Recent evidence indicates that factor VIIa may participate in the intrinsic clotting pathway as well.
Bovine factor VII has been isolated and characterized (Kisiel and Davie, "Biochemistry" 14 (22): 4928-4934 [1975]). The factor VII purified by these authors was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate, exhibited less than 0.001 Ortho units of prothrombin/ml and undetectable levels of factors IX and X when assayed at 0.5 mg protein/ml concentration. Since Factor VII activity losses are high at low protein concentrations, 1 mg/ml of bovine serum albumin was included in factor VII diluents as a stabilizer.
Bovine factor VII is a single-chain glycoprotein of approximately 50,000 daltons. It is converted to a two chain disulfide-linked protein by factor Xa in the presence of calcium ions and phospholipids, or by thrombin or factor XIIa without cofactors. An increase in factor VII activity of about 10 to 100 fold as measured in a one-stage coagulation assay is associated with the conversion to the two chain form.
Human factor VII has also been purified and characterized (Broze and Majeurs, "J. Biol. Chem." 1242-1247 [1780]. This method yields factor VII which, like the bovine product obtained by Kisiel and Davie, was without detectable activity by coagulation assay and did not contain detectable factors X, IX or II. The final preparation contained 2.3 units of factor VII/.mu.g of protein. The human factor VII is 55-85% homologous with the bovine protein and shows many other characteristics in common with bovine factor VII. Like bovine factor VIIa, it is converted to a two chain form upon activation, apparently via proteolytic cleavage. The activity increase upon activation to the two chain from is 20-25 fold. This two chain form will be synonymous hereinafter with factor VIIa.
Factor VIIa previously has not been employed to the applicant's knowledge to treat patients with clotting inhibitors or deficiencies. However, factor VIIa is known to be present in activated prothrombin complex (PCC), a complex mixture of activated and unactivated clotting enzymes used to treat patients having inhibitors of factor VIII.
The therapeutically effective substances in activated prothrombin complexes have been speculated to be one or more of factors IXa, Xa, XIa, XIIa, VIIa, Xa dimer, Xa-prothrombin intermediate binary complex, "large quantities" of factor VII, a "modified form" of factor VII, factor VIII inibitor bypassing activity (FEIBA), platelet factor X activator and, to a small degree, thrombin. The overriding theme espoused by the art, however, is that an incomplete understanding exists of the in vivo mechanism of action for activated PCC, and therefore the art has been able to only speculate as to the effective in vivo hemostatic agent or agents in these compositions.
The therapeutic use of activated PCC would be improved by purifying the effective hemostatic agent. This would result in the administration of less extraneous protein to the patient. In addition, the economics of protein fractionation would be enhanced by diverting the unwanted residue from the purification to the manufacture of other therapeutic products.
Accordingly, it is an object of this invention to identify a hemostatic agent in activated PCC that can be isolated and used alone for the therapy of blood clotting deficiencies.
It is a further object to identify such a hemostatic agent having a low potential for adverse thrombosis such as disseminated intravascular coagulation.
It is an additional object to identify such an agent which can be used so as to not stimulate patients to generate clotting factor inhibitors.
These and other objects of the invention will be apparent from the specification as a whole.